The development and institution of stem cell and tissue engineering methodologies hold the potential to target many of the most significant cardiovascular diseases and have a dramatic impact on the treatment of one of the leading causes of death in the United States healthcare system. The current proposal is a collaborative effort between Athersys, Inc. and the University of Minnesota Stem Cell Institute within their cell therapy program. This project has a main focus on exploiting the Multipotent Adult Progenitor Cell (MAPC) technology as a novel platform for the in-vitro generation of cardiomyocytes for potential application in allogeneic cell therapy in heart disease. The overall objective of this proposal is to synergize the small molecule compound high-throughput screening (HTS) capability at Athersys with the MAPC platform. The working hypothesis is that this provides a unique opportunity to identify factors and pathways that can initiate specific and controlled differentiation of MAPCs into cardiomyocytes or progenitors. The Phase I effort will focus on the use of MAPCs acquired from transgenic mouse models in which the green fluorescent protein (GFP) reporter is specifically expressed in cardiac muscle through the activity of cardiac muscle specific promoter sequences. MAPCs will be isolated from these transgenic mice and expanded for use in HTS assays that measure the induction of GFP fluorescence as a quantitative read-out for differentiation of MAPCs into the cardiomyocyte lineage. The two specific aims in Phase I are; 1) to isolate and expand MAPC from transgenic mice with cardiac muscle specific expression of GFP, and; 2) to establish a sensitive and specific assay for quantification of MAPC differentiation into the cardiomyocyte lineage based on GFP fluorescence. The ultimate ambition of this proposal is to establish the conditions for ex vivo production of cardiomyocyte progenitors from mouse as well as human MAPCs by the end of Phase II. This will provide clinically and commercially relevant new sources for cardiac muscle cells, which are in great demand for in vitro toxicology studies as a near term product, or for the further development of allogeneic cellular therapies for heart disease.